The long-term goal of the proposed research is to elucidate the structure- function relationship of the mammalian Na.K-ATPase, with respect to both catalytic activity and cardiac glycoside binding. The specific aims of this proposal are to identify amino residues that are important for enzyme function in 1) the H7/h8 extracellular alpha subunit domain, which is postulated to be involved in both Na+/K+ and cardiac glycoside interaction; 2) the highly charged NH2-terminal alpha domain, which appears to be involved in K= deocclusion; and 3) to identify domains and residues that are involved directly and indirectly in the enzyme's interaction with ouabain and related drugs. The experimental approach for Aims 1 & 2 is to use site-directed mutagenesis and expression of the ouabain-insensitive rat alpha1 isozyme in HeLa cells, which contain an endogenous ouabain-sensitive isoform of Na.K- ATPase. Selection of stable clones with either ouabain or G418 enables us to quickly determine if any mutant enzyme is capable of catalyzing Na+/K+ transport or if it is "lethal". Active mutant enzymes will be functionally characterized with respect to Na.k-ATPase:E-P ratio. I50 for ouabain, 86Rb uptake, and K0.5's for ATP, Na+ and K+. The ability of inactive mutant enzymes (selected with G418) to be phosphorylated from ATP and Pi will also be measured. In addition, some mutant cDNA cassettes will be cloned into a modified rat alpha 1S cDNA, that encodes a ouabain-sensitive Na,K-ATPase (KD=1.57 nM), and expressed in ouabain-insensitive NIH/3T3 cells. This allows [3H] ouabain binding to be used as a tool for measuring the affinity of the mutant enzymes for ouabain, as well as the enzyme's affinity for those ligands that regulate ouabain binding (e.g., ATP. Pi, vanadate.Na+, K+ and Mg++). The comparison of functional parameters between wild type and mutant enzymes will define the roles that alpha subunit domains and specific residues play in th function of the Na.K-ATPase. These studies are currently ongoing in the applicant's laboratory. Good progress on specific aims 1 and 2 has already been achieved and initial studies on aim 3 are underway.